Researchers may use microscopy imaging systems during high-content screenings (HCS) to obtain images of microscopy samples. A sample holder—e.g., a microtitre plate, slide, dish, etc.—may support the microscopy samples during the screening process. Microscopy imaging systems may include an adjustable objective to produce the images of the microscopy samples. The position of the objective relative to the sample holder may be adjusted to bring the microscopy samples into focus. In order to produce an in focus image, the objective should be positioned at an appropriate distance from the focal plane for a microscopy sample. The distance between the objective and the focal plane for a microscopy sample may be referred to as the focal position of the objective.
The microscopy samples may reside at various measurement locations (e.g., wells) on the upper surface of the sample holder. The upper surface of the sample holder may thus correspond to the focal plane for a microscopy sample. Accordingly, the objective of the microscopy imaging system may be positioned at a focal position relative to the bottom of the sample holder in order to obtain an in focus image of a microscopy sample. Variations in the thickness or curvature of the sample holder, however, may prevent accurate focus over a range of measurement locations. As a result, the focal position of the objective may need to be corrected at each measurement location in order to obtain respective in focus images for all measurement locations. Because high content screenings may image hundreds or thousands of measurement samples, some microscopy imaging systems may be configured to automatically perform focus maintenance at each measurement location.
Some example microscopy imaging systems may automatically adjust the position of the objective using laser beams and a linear detector. In these example types of microscopy imaging systems, an off-axis laser beam is directed towards the lower surface of the sample holder where the beam is then reflected off the lower surface, back through the objective, and onto the linear detector. Because the laser beam directed towards the sample holder is off-axis, the position of the reflected laser beam on the linear detector thus corresponds to the position of the objective relative to the lower surface of the sample holder. If the objective moves to a new position relative to the lower surface of the sample holder, then the reflected laser beam will also move to a new position on the linear detector. The objective, in this instance, may be manually positioned at a focal position, and the corresponding position of the reflected laser beam on the linear detector may be recorded. Whenever a new measurement location is imaged, the current position of the reflected laser beam on the linear detector may be compared to the recorded position on the linear detector. If the current position of the reflected laser beam differs from the recorded position, then the position of the objective relative to the lower surface of the sample holder may be adjusted until the current position of the reflected laser beam on the linear detector matches the recorded position. The objective may also be offset from the focal position where the offset corresponds to the thickness of the sample holder. Offsetting by thickness, however, may not be accurate enough, in some circumstances, to achieve an optimal focus position unless additional focus information is provided by an image autofocus or a manual focus.
In these example types of microscopy imaging systems, however, multiple laser beams may be reflected off the lower surface, the top surface, or both the lower surface and the top surface of the sample holder. Reflected laser beams may not be distinguishable from one another. Accordingly, the reflected laser beam striking the linear detector may have originated at the upper surface or the lower surface of the sample holder. Additionally, multiple reflected laser beams may strike the linear detector in this example. As a result, adjustment of the objective may be inaccurate causing images of the microscopy samples to be out of focus. The problem of multiple reflections may be particularly pronounced where the microscopy system seeks to identify the top surface of the sample holder from below.
Other example microscopy imaging systems may also use laser beams to automatically adjust the position of the objective. In these other example types of microscopy imaging systems, the focal position for the objective is determined based on an observed peak laser beam intensity. In this example, the focal position for the objective is determined to be the position that results in the most intense (i.e., brightest or smallest) laser beam reflection. Because both the lower surface and the upper surface will reflect the laser beam, however, a search procedure may be necessary to distinguish the location of the lower surface from the upper surface of the sample holder. The objective is positioned at decreasing distances relative to the sample holder, and reflected laser beam intensities are observed at each position. Accordingly, a peak laser beam intensity may result at both the lower surface and the upper surface of the sample holder. In this example, the first peak laser beam intensity typically corresponds to the lower surface of the sample holder, and the second peak laser beam intensity typically corresponds to the upper surface of the sample holder. The position of the objective corresponding to the second peak laser beam intensity is thus identified as the focal position for the microscopy sample.
In these other example types of microscopy imaging systems, searching for both the lower surface and the upper surface of the sample holder can be time-consuming where the sample holder includes hundreds or thousands of measurement locations. These other example types of microscopy imaging systems may be configured to attempt to search only for the upper surface. Reflections from the lower surface may, however, interfere with reflections from the upper surface of the sample holder thus increasing the likelihood that the search for the upper surface will fail. If the search for the upper surface fails, these other types of example microscopy imaging systems may resort to searching for both the lower surface and the upper surface of the sample holder thus increasing the processing time of the imaging procedure.
Therefore, improved systems and methods for determining a focal position of an objective of a high content screening microscopy imaging system are needed.